Free divalent cation regulates such cell functions as growth and hormone response. While Ca++ has received much attention, free Mg++ has distinct roles neither inhibited nor mimicked by Ca++. This laboratory has described a Mg++ transport system in the murine S49 lymphoma cell which is inhibited by Beta-adrenergic receptor stimulation; however, this inhibition is not mediated by cyclic AMP. Thus, Mg++ transport may be an integral function of the hormone receptor-adenylate cyclase complex. We have now demonstrated two distinct sites for free Mg++ regulation on the receptor-cyclase complex, both accessible only from the cytoplasmic membrane face. Further, the Mg++ transport system transports Mg++ into a subcytoplasmic Mg++ compartment which is less than 3% of total cell Mg++, is totally cytoplasmic, and does not exchange with the remainder of cell Mg++ for at least 2 hours after entry into the cell. This pool of Mg++ is hypothesized to be involved in chronic regulation of adenylate cyclase response to hormonal stimulation. To test this working hypothesis, a comprehensive study of the role of Mg++ in the regulation of cell response to hormonal stimulation is outlined. a) Mg++ transport and compartmentation will be correlated with the characteristics of Mg++ regulation of adenylate cyclase in S49 cells and three other cell lines. The latter have been selected because they may transport or compartment Mg++ differently than the S49 cell. b) The effect of hormone on free intracellular [Mg++] will be measured using 31p-NMR and cell-permeable fluorescent probes selective for Mg++. Conversely, the effect of altering cell [Mg++] on hormone response will be tested using ionophores with selectivity for Mg++ over Ca++. c) The mechanism of hormone-sensitive Mg++ transport will be investigated in sealed S49 cell membrane vesicles as a prelude to the reconstitution of hormone-sensitive Mg++ transport in liposomes. d) Variants of S49 lymphoma cells resistant to the cytocidal effects of low growth medium [Mg++] will be isolated to facilitate study of the mechanism of Mg++ transport.